Spatial regulation of exocytosis is essential for establishing and maintaining cell surface polarity, a property vital to the correct function of epithelia. Failure to properly sort and deliver specific plasma membrane proteins to the correct surface of epithelial cells is a common feature of human metabolic diseases and cancers. Despite much progress in understanding the molecular mechanisms of protein sorting, major questions persist about how post-Golgi transport vesicles ferry apical and basolateral cargo to the correct site of fusion with the plasma membrane. The long-term goal of our research is to understand how environmental spatial cues, such as cell-cell adhesion, signal to the cytoskeleton and secretory apparatus to organize polarized trafficking pathways in epithelial cells. Based upon results of preliminary studies, a working hypothesis has been developed. Our central hypothesis is that Ral GTPases are activated upon initiation of E-cadherin-mediated cell adhesion, and that they regulate Exocyst functions in a spatio-temporal manner during the formation of specialized membrane domains-including the apical junctional complex, desmosomes and primary cilia. Our working model is that Ral GTPases regulate different steps in Exocyst-dependent trafficking, through sequential interactions with the Sec5 and Exo84 subunits. These interactions, in turn, drive an association/dissociation cycle between the Exocyst and "accessory factors" that either target Exocyst complexes to different subcellular sites or otherwise prepare them for specific functions at those sites. Finally, we propose that at least two different Exocyst-related complexes are recruited to nascent sites of intercellular contact, and subsequently move apart to facilitate the development of different types of intercellular junctions. The specific aims of this study are: 1) to investigate the function of Ral-Exocyst complexes in modulating epithelial polarization;2) to determine how Sec6-Munc18 interactions control polarized exocytosis;and 3) to define how Sec6 is targeted to the AJC and determine whether multiple Exocyst-related complexes have distinct functions in intercellular junction formation. Collectively, the studies we propose in this application seek to uncover details of a mechanism that links an extracellular event, namely cell-cell adhesion, to activation of a signaling pathway that promotes polarized membrane trafficking during the establishment of epithelial polarity. Given that the molecules on which we will focus-specifically the Exocyst and Ral GTPases-are conserved across metazoans, we believe that studying how they collaborate to establish and maintain distinct junctional complexes in epithelial cells (e.g. the AJCs and desmosomes) will give us important insight into mechanisms that regulate the assembly of plasma membrane protein complexes in many other cell types. Thus, the significance of this work is that it will guide our thinking when considering therapies for a variety of human diseases in which intracellular transport and the assembly of membrane proteins is impaired. PUBLIC HEALTH RELEVANCE: In order for our epithelial cells to protect us from harmful agents that cause disease, they must organize components of the plasma membrane into structurally and functionally distinct domains. Failure to do so results in devastating human diseases, including hypercholesterolemia, diabetes, and polycystic kidney disease. This proposal will investigate molecular mechanisms responsible for guiding vesicles carrying receptors to the right site in the cell for insertion into the plasma membrane, and extend our understanding of the basis for abnormalities in membrane protein organization characteristic of epithelial diseases.